Preparation of electrolyte gradient gel experiment
Preparation of electrolyte gradient gel experiment
Sheen and Seed (1980) created a clever and simple improvement by utilizing different concentrations of electrophoresis buffer at the top and bottom to create an ionic gradient in the gel, while the gel itself is at a constant concentration. Using this method, the total number of nucleotides that can be read from a single gel can be increased by about 30%. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Preparation of electrolyte gradient gel experiment
Materials and Instruments
This method includes all of the items in Scheme 8 plus: Formamide (100%) Ion Gradient Gel Sodium Acetate Move makings For more product details, please visit Aladdin Scientific website.
This method includes all items in Option 8, plus:
Formamide (100%)
The ionic gradient gel is prepared with 1XTBE with or without formamide. For polyacrylamide gels containing formamide, see Scheme 9.
Sodium acetate (3 mol/L, pH 7.0)
Methods
1. Follow Steps 1 to 8 of Scheme 8.
2. Line the work area of the laboratory table with plastic-lined protective paper.
3. Prepare denaturing polyacrylamide gel according to protocol 8 or 9.
4. Place the gel in the electrophoresis apparatus. Add 0.5 XTBE buffer to the upper tank and 1 part 3mol/L sodium acetate and 2 parts 1XTBE to the lower tank. follow protocol 11.
During electrophoresis, the movement of the indicator dye gradually slows down and almost stops when the bromophenol blue reaches the bottom. Generally, electrophoresis was continued until xylene blue was only 5-10 cm from the bottom and bromophenol blue reached the bottom.
Delaying the addition of 3mol/L sodium acetate can adjust the time of compression of the DNA bands. Start electrophoresis with 1XTBE in both the top and bottom tanks; at the appropriate time, add half the volume of 3 sodium acetate to the bottom tank. As a rule of thumb, this compresses the different regions of the bands and reads the most bands.